1. Field of the Invention
This invention relates to a novel immuno-dye reagent and an assay for the detection of endotoxin.
2. Description of the Background Art
Lipopolysaccharide (LPS), an endotoxin, is a major outer membrane component of the cell walls of all Gram-negative bacteria. It is composed of a polysaccharide chain of repeating oligosaccharide units (the O-antigen polysaccharide), a middle core polysaccharide and a lipid-containing moiety (lipid A) by which the LPS macromolecule is anchored in the outer membrane. The O-antigen polysaccharide is composed of repeating oligosaccharide, specific to the species and the strain of the bacteria and the core polysaccharide consists of 11 or less monosaccharide units including three 2-keto-3-deoxyoctonate (KDO). Lipid A usually consists of a phosphorylated glucosamine disaccharide substituted with ester- and amide-linked fatty acids (Brade and Galanos, 1982; Luderitz et al., 1973; Galanos et al., 1985).
The lipid A moiety is embedded in the bacterial membrane while the polysaccharide chain is extruding out of the membrane into hydrophilic surroundings. When LPS is released from bacteria into the aqueous medium, it forms micelles (in the shape of ribbons, disks and lamellae, etc.) and vesicles of bilayer structure with the hydrophobic portion (fatty acid chains) buried and the hydrophilic portion (the phosphate groups, KDO and the polysaccharide chain) exposed to the aqueous medium.
Endotoxins are tenacious contaminants of aqueous and physiological solutions. Endotoxins contaminate medicines and medical devices during preparation processes. They are comparatively heat stable and have a remarkable pyrogenic action (Shenep, J.L. and D.A. Morgan. "Kinetics of Endotoxin Release During Antibiotic Therapy for Experimental Gram-negative Bacterial Sepsis," J Infect Dis. 150:380-388, 1984). Bacterial endotoxins have the ability to cause various biological reactions in man and animals such as fever, leukopenia, disseminated intravascular coagulation, endotoxin shock, and death (Luderitz, O., Galanos, C., Lehmann, V., Mayer, H., Rietschel, E.T., and J. Wechersser. "Chemical Structure and Biological Activities of Lipid A's from Various Bacterial Families," Natur wissenschaften. 65:578-585,1978).
The death rate from Gram-negative bacteremia and endotoxin remains unacceptably high (30-40%) despite modern advances in antibiotics, pressors and intensive care (Lachman, E., Pitsoe, E., and S.L. Gaffin. "Anti-lipopolysaccharide Immunotherapy in Management of Septic Shock of Obstetric and Gynecological Origin," Lancet, I:981-983, 1984). Lipid A is considered to be the toxic moiety in LPS which participates in the pathogenesis of septic shock exhibiting the pathological effects such as pyrogenicity, activation of coagulation factors, renal failure and hepatocytotoxicity.
The structural diversity of LPS (e.g. the polysaccharide chain) from different origins and its amphipathic property have posed serious difficulties to the prospect of its broad spectrum recognition by immunoglobulins that constitute a critical immunologic defense mechanism among others.
The importance of developing an assay for the detection of endotoxin is illustrated in the case of bacterial meningitis. Despite the availability of potent antibiotics, bacterial meningitis remains an important source of morbidity and mortality (3-30%) in newborns and young children around the world (Marks, M.I. "Bacterial Meningitis in Infants and Children," Infection, 12:s52-s55, 1984). It is caused by a variety of Gram-negative and Gram-positive organisms as well as viruses. The principal bacterial etiologic agents are Haemophilus influenzae. Streptococcus pneumoniae, Staphylococcus aureus. Escherichia coli and Neisseria meningitides. Meningitis presents a medical emergency that requires early medical therapy to prevent death, serious neurologic defects or impaired learning. Speed in establishing the diagnosis and initiating specific therapy is therefore very essential (Klein, J.O. "Recent Advances in Management of Bacterial Meningitis in Neonates," Infection, 12:s44-s51, 1984). The recent localized outbreaks of the disease in the U.S. and other regions around the world further suggest the need to improve surveillance (Center for Disease Control. "Summary of Notifiable Diseases, United States, 1987." 1988.).
At present, bacterial meningitis has been detected by countercurrent-immunoelectrophoresis (CIE) of specific polysaccharide antigens in CSF (Kaplan, S.L. and R.D. Feigin. "Rapid Identification of the Invading Microorganism," Pediatr. Clin. North Am., 27:783-803, 1980), Limulus amebocyte lysate assays (Nachum, R., Lipsey, A., and S.E. Siegel. "Rapid Detection of Gram-negative Bacterial Meningitis by the Limulus Lysate Test," New Engl. J. Med., 289:931-934, 1973), standard bacteriological methods, organisms-specific latex agglutination (Severin, W.P.J. "Latex Agglutination in the Diagnosis of Meningicoccal Meningitis," J. Clin. Pathol., 25:1079-1082, 1972) and enzyme-linked immunosorbent assay (ELISA) (Adams, L. B., Henk, M.C., and R.J. Sieberling. "Detection of Vibrio cholerae with Monoclonal Antibodies Specific for Serovar 01 Lipopolysaccharide," J. Clin. Micro., 26:1801-1809, 1988). CIE is capable of detecting some of the infecting organisms, but is limited by the problems of non-specificity, lack of broad specificity and limited sensitivity of polyclonally produced antibodies. Furthermore, it is highly impractical for large numbers of sample or field conditions. Standard bacteriological assay by CSF culture, in contrast, is very reliable but requires prolonged incubation time before results can be available in at least 18 to 24 hours. This is a major disadvantage when prompt detection has been shown to be of great importance to reducing the case fatality rate of meningitis by several investigators (Marks, s52-s55).
The use of the Limulus amoebocyte lysate assay (LAL) for detecting and quantitating endotoxins is well established but, when applied to blood samples, it is restricted because of the presence of various factors in blood that interfere with the LAL endotoxin reaction (Stumacher, R.J., Kovnat, M.J., and W.R. McCabe. "Limitations of the Usefulness of the Limulus Assay for Endotoxin," N. Engl. J. Med., 288:1261-1264, 1973). Also most LAL test methods include periods of one hour or more.
Under many circumstances in both industry and clinical research situations, i.e. pharmaceutical industry, it is highly desirable to be able to identify endotoxin contamination promptly and accurately in non-biological and biological fluids (serum, plasma, cerebrospinal fluid (CSF)). Such a method does not exist at present.